Development of the Pronotal Explanate Margin, a Novel Evolutionary Trait in Tortoise Beetles.

Most tortoise beetles, belonging to the subfamily Cassidinae (Coleoptera: Chrysomelidae), possess distinctive explanate margins, comprising elongations of the pronotum and elytra outer margins. These margins flatten against the ground, serving as a unique defensive mechanism against predators. To understand the developmental and evolutionary origins of explanate margins, we examined the development of the pronotal part of these structures in two tortoise beetle species: Thlaspida biramosa (Boheman) (tribe Cassidini) and Laccoptera nepalensis (Boheman) (tribe Aspidimorphini). Although final (fifth) instar larvae of both species exhibited no external prothoracic structures associated with explanate margins, pupae possessed a plate-shaped structure projecting anterolaterally on their pronotum. This plate-shaped structure was identified as the pupal primordium of the pronotal explanate margin, as the explanate margin emerged from inside the structure during eclosion. In prepupae of T. biramosa, the primordial tissue exhibited three region-specific folding and furrowing patterns, beneath larval cuticles. These epithelial structures expanded within minutes at the onset of pupation, resulting in pupal primordial formation. Thus, pronotal explanate margins originate from pronotal epithelia, with the furrowing and folding patterns of the epithelia shaping pupal and even adult pronotal explanate margins. The presence of morphologically similar pupal pronotal projections in various Cassidinae suggests that the development of pronotal explanate margins is likely common in these beetles.

A window on remarkable cryptic diversity of the Merodon planifacies subgroup (Diptera: Syrphidae) in the Afrotropical Region.

The genus Merodon Meigen (Diptera: Syrphidae) is one of the most species-rich hoverfly genera distributed across the Palaearctic and Afrotropical regions. In the Palaearctic, the genus Merodon boasts 195 described species, while its Afrotropical region pales in comparison, with a mere 17 species documented thus far. As a result of 8 years of fieldwork conducted in the Republic of South Africa, in this paper, we present the description of 11 new species for science with a description of immature stages for 2 species, which increases the diversity of this genus in the Afrotropical region by remarkable 39%. These revelations are based on integrating morphology, molecular analysis (COI gene and 28S rRNA) and geometric morphometry. All described species belong to the Merodon planifacies subgroup, the Merodon desuturinus lineage and, within that, to the Afrotropical Merodon melanocerus group. Additionally, we provide an illustrated key to 15 species belonging to the subgroup, a detailed discussion on relevant taxonomic characters, a morphological diagnosis, a distribution map and clarification of the association between M. capi complex and host plants from the genus Merwilla.

Transcriptomic Analysis of Genes Associated with Stinger Development at Different Life Stages of Apis mellifera.

Stingers, evolved from ovipositors, are an important defense organ for the Apidae, Vespidae, and Formicidae species. However, the molecular mechanism of stinger development remains unclear. Here, we show that the earliest time point for the appearance of stingers in Apis mellifera is at the 1-day-old worker pupal stage based on morphological observations and anatomy from the pre-pupal to adult stages. To discover the genes related to stinger development, we first comprehensively compared the stinger transcriptome at different stages and screened 1282, 186, and 166 highly expressed genes in the stingers of 1- and 5-day-old worker pupae and newly emerged worker bees (NEBs), respectively, then identified 25 DEGs involved in the early stage of stinger development. We found that Dll was a key candidate gene in the early development of A. mellifera stingers by combining analyses of the protein-protein interaction network and spatiotemporal expression patterns. An RNAi experiment showed that about 20% of individuals exhibited tip bending in the piercing parts of their stingers in the Dll-dsRNA-treated group, with the morphology presenting as side-side or front-back tip bending. This indicates that Dll plays a vital role in the early development of A. mellifera stingers. Together, our study provides insight into the molecular mechanism of Hymenoptera stinger development and an inspiration for the molecular breeding of gentle honeybee species with stinger abnormalities.

Evaluating the effects of short-term low temperature on the growth and development of Trichopria drosophilae based on the age-stage two-sex life table.

BACKGROUND: The effects of low temperatures on parasitic wasps are crucial for maintaining farmland biodiversity and enhancing biological control, especially given the implications of global warming and frequent extreme cold events. METHODS: We studied the effects of different low temperatures (-8 ± 1 °C, -4 ± 1 °C, 0 ± 1 °C, 4 ± 1 °C, and 8 ± 1 °C) on the mating frequency and duration of male adults of Trichopria drosophilae and the number of pupae beaten by female adults, and constructed the age-stage two-sex life table of T. drosophilae. RESULTS: This study found that male T. drosophilae adults exposed to low temperatures for 12 h significantly altered their mating behavior, peaking between 15:00 and 17:00. As the temperature dropped during the exposure, both the mating frequency of T. drosophilae and the duration of pupal beating were affected. The survival rate of female adults dropped from 39.55% at 8 °C to just 21.17% at -8 °C. Low-temperature treatment shortened the development period and lifespan for T. drosophilae adults. They developed 4.71 days faster and had a total lifespan that was 10.66 days shorter than those in the control group after being exposed to -8 °C. Furthermore, the average number of eggs laid by females at -8 °C was 4.46 less than that at 8 °C and 6.16 less than that in the control group, which laid an average of 21.55 eggs. The net reproductive rate (R0) of T. drosophilae decreased with lower temperatures, reaching a low of 23.64 at -8 °C. Conversely, the intrinsic growth rate (rm) actually increased as temperatures dropped, with the lowest value being 0.21 at -8 °C. CONCLUSIONS: The findings indicate that short-term exposure to low temperatures hampers the growth and population increase of T. drosophilae, thereby reducing their effectiveness as biological control agents.

Knockdown of TcGluCl leads to the premature pupation of Tribolium castaneum larvae possibly by influencing the calcium-mediating hormone homeostasis.

The glutamate-gated chloride channels (GluCls) are widely existed in the neural and nonneural tissues of invertebrate. In addition to play important roles in signal transduction, the GluCls also showed multiple physiological functions in insects such as participate in the juvenile hormone synthesis. In the present study, the potential roles of TcGluCl in growth and development of the red flour beetle Tribolium castaneum were explored. Knockdown of TcGluCl showed no effects on the survivability, weight growth, final pupation rate, eclosion and fecundity of T. castaneum, whereas resulted in the significant premature pupation of larvae. Inhibition of TcGluCl expression significantly changed the levels of juvenile hormone and ecdysone as well as the expressions of hormone biosynthetic genes. The increased ecdysone level and decreased juvenile hormone level were observed at the late stage of dsGluCl-treated larvae. Knockdown of TcGluCl significantly reduced the expressions of TcSTIM1 and TcOrai1, which were the primary proteins in store-operated calcium entry (SOCE) mediated Ca2+ influx mechanism. Whilst the L-glutamic acid treatment led to the increased TcOrai1 expression in T. castaneum. These findings suggested that knockdown of TcGluCl increased the ecdysone level and contributed to the premature pupation of larvae, which might be due to the reduced Ca2+ influx caused by the decreased expressions of TcSTIM1 and TcOrai1. These studies provide novel insights on the function of GluCls in insects.

The role of neuropeptide prothoracicotropic hormone (PTTH) - Torso in pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms.

The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms.

Larval development of a parasitoid depends on host ecdysteroids.

Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20 E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20 E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20 E signaling and the subsequent 2nd-instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control.

Soluble Guanylate Cyclase α1 Gene Influences Egg-Laying Amount and Hatching Rate in Bombyx mori.

Soluble guanylate cyclase (sGC) serves as a receptor of nitric oxide (NO) and is the core metalloenzyme in the NO signal transduction pathway. sGC plays a key role in the NO-cGMP signal transduction pathway and participates in various physiological processes, including cell differentiation, neuron transmission, and internal environment homeostasis. sGC consists of two subunits, α and ß, each subunit containing multiple isoforms. In this study, we cloned and analyzed the sGC-α1 gene in the silkworm Bombyx mori (BmsGC-α1). The BmsGC-α1 gene was expressed highest at the pupal stages. The highest BmsGC-α1 mRNA expression was observed in the head of fifth instar larvae and in fat body during the wandering stage of B. mori. Furthermore, we observed that feeding fifth instar larvae with thyroid hormone and nitroglycerin induced the expression of the BmsGC-α1 gene. Injection of BmsGC-α1 siRNA into silkworms at the prepupal stage resulted in a significant decrease in BmsGC-α1 expression levels at 48 and 72 h postinjection. After silencing BmsGC-α1, both the egg-laying amount and hatching rate of silkworm eggs were significantly reduced compared to the control group. These results suggest that BmsGC-α1 plays an important role in regulating the reproductive system of silkworms. This finding enhances our understanding of the functional diversity of sGC in insects.

Phytosanitary cold treatment of cherry tomatoes infested with Bactrocera dorsalis, Zeugodacus cucurbitae, and Zeugodacus tau (Diptera: Tephritidae).

Fruit flies attack numerous crops, including cherry tomatoes (Solanum lycopersicum var. cerasiforme). The potential presence of the immature stages of fruit fly species inside tomatoes during export hinders their international market access. Therefore, phytosanitary treatment must be performed before export to prevent fruit fly species from entering countries where they are not naturally found. We developed a phytosanitary cold disinfestation treatment protocol to eliminate oriental fruit fly (Bactrocera dorsalis Hendel), melon fly (Zeugodacus cucurbitae Coquillett), and pumpkin fruit fly (Zeugodacus tau Walker) concealed inside cherry tomatoes without causing critical damage to the fruit. We determined that the third instar of Z. cucurbitae exhibited the highest cold tolerance among the various development stages of the three fruit fly species. Thus, we performed a small-scale disinfestation test on Z. cucurbitae in two cultivars of tomato. We achieved complete disinfestation after 15 days of cold treatment at 1°C-1.5°C. The confirmatory test revealed the elimination of more than 80,000 treated third instar of Z. cucurbitae in each tomato variety. The developed phytosanitary cold treatment allows the tomatoes to retain their commercial value. This study provides a standard phytosanitary cold treatment protocol for cherry tomatoes, ensuring the disinfestation of fruit flies before their export to international markets.

Modeling long-term, stage-structured dynamics of Tribolium castaneum (Coleoptera: Tenebrionidae) at food facilities with and without two types of long-lasting insecticide-incorporated netting.

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a cosmopolitan and destructive external-infesting pest at many food facilities. The use of deltamethrin- and α-cypermethrin-incorporated long-lasting insecticide-incorporated netting (LLIN) has shown incredible promise for the management of stored product insects. However, it is unknown how LLIN deployed within food facilities may affect the long-term population dynamics of T. castaneum compared to populations where no LLIN is present. Exposure to LLIN has been shown to affect mortality in the current generation and decrease progeny production in the subsequent generation. Thus, we modeled the long-term population dynamics of T. castaneum at food facilities over 15 generations by incorporating realistic estimates for mortality and progeny reduction after contact with LLIN compared to baseline growth by the species. We parameterized the model with estimates from the literature and used a four-stage structured population (eggs, larvae, pupae, and adults). The model was implemented using the package popbio in R. Our models suggest that deploying LLIN led to significant population reductions based on the estimates of mortality and progeny reduction from prior work, whereas the baseline model exhibited exponential population growth. In addition, there were differences in the frequencies of each life stage under each scenario modeled. As a result, it appears deploying LLIN may contribute to the local extirpation of T. castaneum within as few as 15 generations. Our work contributes to a growing literature about the effectiveness of incorporating LLIN into existing pest management programs for managing stored product insects in food facilities.

Life table study of Rachiplusia nu (Lepidoptera: Noctuidae) on different food sources and artificial diet.

Rachiplusia nu Guenée is a polyphagous species able to develop on several cultivated and non-cultivated host plants. However, basic life history information about this pest on hosts is scarce. In this study, R. nu larvae did not survive on leaves of non-Bt corn, wheat, Bt cotton that expresses proteins Cry1Ac+Cry2Ab2 or on Intacta2 Xtend soybean that expresses the Cry1A.105/Cry2Ab2/Cry1Ac proteins. Rachiplusia nu showed a viable egg-to-adult biological cycle (54%-66.3%) on non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip hosts, similar to larvae raised on the artificial diet. In addition, R. nu was unable to complete larval development on non-Bt cotton, and only 45.2% of R. nu larvae reached the pupal stage when fed leaves of intacta RR2 PRO soybean that expresses the Cry1Ac protein. Larval and pupal mass of surviving insects on Cry1Ac soybean leaves were also lower (larval: 0.104 g; pupal: 0.099 g) in relation to other food sources (larval: 0.165-0.189 g; pupal: 0.173-0.192 g). The total fecundity of R. nu on Cry1Ac soybean leaves was ≈65% lower in relation to other food sources. This fact caused ≈60% the net reproductive rate (Ro) and intrinsic rate of increase (rm) when compared to other food sources. Our findings indicate that the Cry1Ac soybean negatively affects the biological parameters of R. nu. Non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip are viable food sources for the survival and development of R. nu.

The irregular developmental duration mainly caused by the broad-complex in Chilo suppressalis.

Chilo suppressalis, a critical rice stem borer pest, poses significant challenges to rice production due to its overlapping generations and irregular developmental duration. These characteristics complicate pest management strategies. According to the dynamic analysis of the overwintering adults of C. suppressalis in fields, it indicates that the phenomenon of irregular development of C. suppressalis exists widely and continuously. This study delves into the potential role of the Broad-Complex (Br-C) gene in the developmental duration of C. suppressalis. Four isoforms of Br-C, named CsBr-C Z1, CsBr-C Z2, CsBr-C Z4, and CsBr-C Z7, were identified. After CsBr-Cs RNAi, the duration of larva development spans extended obviously. And, the average developmental duration of dsCsBr-Cs feeding individuals increased obviously. Meanwhile, the average developmental duration of the dsCsBr-C Z2 feeding group was the longest among all the RNAi groups. After dsCsBr-Cs feeding continuously, individuals pupated at different instars changed obviously: the proportion of individuals pupated at the 5th instar decreased and pupated at the 7th instar or higher increased significantly. Moreover, the pupation rate of dsCsBr-Cs (except dsCsBr-C Z7) were significantly lower than that of dsGFP. The same results were obtained from the mutagenesis in CsBr-C genes mediated by CRISPR/Cas9. The average developmental duration of CsBr-Cs knockout individuals was significantly prolonged. And, the instar of pupation in knockout individuals was also delayed significantly. In conclusion, this work showed that CsBr-Cs played a crucial role in pupal commitment and affected the developmental duration of C. suppressalis significantly.

Gut fungal diversity across different life stages of the onion fly Delia antiqua.

A significant number of microorganisms inhabit the intestinal tract or the body surface of insects. While the majority of research on insect microbiome interaction has mainly focused on bacteria, of late multiple studies have been acknowledging the importance of fungi and have started reporting the fungal communities as well. In this study, high-throughput sequencing was used to compare the diversity of intestinal fungi in Delia antiqua (Diptera: Anthomyiidae) at different growth stages, and effect of differential fungi between adjacent life stages on the growth and development of D. antiqua was investigated. The results showed that there were significant differences in the α and ß diversity of gut fungal communities between two adjacent growth stages. Among the dominant fungi, genera Penicillium and Meyerozyma and family Cordycipitaceae had higher abundances. Cordycipitaceae was mainly enriched in the pupal and adult (male and female) stages, Penicillium was mainly enriched in the pupal, 2nd instar and 3rd instar larval stages, and Meyerozyma was enriched in the pupal stage. Only three fungal species were found to differ between two adjacent growth stages. These three fungal species including Fusarium oxysporum, Meyerozyma guilliermondii and Penicillium roqueforti generally inhibited the growth and development of D. antiqua, with only P. roqueforti promoting the growth and development of female insects. This study will provide theoretical support for the search for new pathogenic microorganisms for other fly pests control and the development of new biological control strategies for fly pests.

The transcription factor RUNT-like regulates pupal cuticle development via promoting a pupal cuticle protein transcription.

Holometabolous insects undergo morphological remodeling from larvae to pupae and to adults with typical changes in the cuticle; however, the mechanism is unclear. Using the lepidopteran agricultural insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the transcription factor RUNT-like (encoded by Runt-like) regulates the development of the pupal cuticle via promoting a pupal cuticle protein gene (HaPcp) expression. The HaPcp was highly expressed in the epidermis and wing during metamorphosis and was found being involved in pupal cuticle development by RNA interference (RNAi) analysis in larvae. Runt-like was also strongly upregulated in the epidermis and wing during metamorphosis. Knockdown of Runt-like produced similar phenomena, a failure of abdomen yellow envelope and wing formation, to those following HaPcp knockdown. The insect molting hormone 20-hydroxyecdysonen (20E) upregulated HaPcp transcription via RUNT-like. 20E upregulated Runt-like transcription via nuclear receptor EcR and the transcription factor FOXO. Together, RUNT-like and HaPCP are involved in pupal cuticle development during metamorphosis under 20E regulation.

Rapid growth and the evolution of complete metamorphosis in insects.

More than 50% of all animal species are insects that undergo complete metamorphosis. The key innovation of these holometabolous insects is a pupal stage between the larva and adult when most structures are completely rebuilt. Why this extreme lifestyle evolved is unclear. Here, we test the hypothesis that a trade-off between growth and differentiation explains the evolution of this novelty. Using a comparative approach, we find that holometabolous insects grow much faster than hemimetabolous insects. Using a theoretical model, we then show how holometaboly evolves under a growth-differentiation trade-off and identify conditions under which such temporal decoupling of growth and differentiation is favored. Our work supports the notion that the holometabolous life history evolved to remove developmental constraints on fast growth, primarily under high mortality.

Juvenile responses to immune challenges are not carried through to subsequent life stages in an insect.

Environmental variability can significantly impact individual survival and reproduction. Meanwhile, high population densities can lead to resource scarcity and increased exposure to parasites and pathogens. Studies with insects can offer valuable insights into eco-immunology, allowing us to explore the connections between these variables. Here we use the moth Anticarsia gemmatalis to examine how increases in population density and immunological challenge during the larval stage shape its investment in immune defence and reproduction. Larvae reared at a high population density exhibited greater lytic activity against bacteria compared to those reared at low density, whilst bacterial challenge (i.e. bacteria-immersed needles) also increased lytic activity. There was no interaction between the variables population density and bacterial challenge, indicating that these are independent. Surprisingly, neither increase in lytic activity carried through to activity in prepupal haemolymph. Rearing of larvae at a high density delayed pupation and decreased pupal weight. The immunological stimulus did not significantly influence pupal development. Lower population density as a larva resulted in greater adult weight, but did not significantly influence lytic activity in the eggs or the number of eggs laid. Negative correlations were found between lytic activity in the eggs and the number of eggs, as well as between adult weight and the number of eggs. Overall, this study demonstrates that high population density and immune challenge trigger increased lytic activity in caterpillars, but this effect is transient, not persisting into later stages. The trade-offs observed, such as delayed pupation and reduced prepupal weights under high density, suggest a balancing act between immune investment and developmental aspects. The findings hint at a short-term adaptive response rather than a sustained strategy. The implications of delayed pupation and smaller adult moths could influence the moth's life history strategy, impacting its role in the ecosystem. Further research tracking larval immune investment and subsequent reproductive success will unveil the evolutionary dynamics of this relationship in changing environments.

Overwintering potential of the Mediterranean fruit fly (Diptera: Tephritidae) in Austria.

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), one of the most important invasive pests of fresh fruits and vegetables from the coastal Mediterranean habitats, is expanding its current geographic distribution to cooler more temperate areas of Europe. Every year since 2010 the fly is detected in the area of Vienna, Austria. However, whether it can establish permanent populations is not known. In this current paper, the capacity of C. capitata to overwinter in Vienna, Austria (48.1° northern latitude) was studied over 2 consecutive winter seasons (2020-2022). Overwintering trials with different life stages (larva, pupa, and adult) of C. capitata were performed in the open field and in the protected environment of a basement without a heating system. Control flies were kept under constant conditions in a climate chamber (25 °C, 60% RH, 14:10 L:D). Our data showed that no life stage of the Mediterranean fruit fly was able to survive the Austrian winter in the open field. However, in the protected environment C. capitata outlived the winter months in all studied life stages at least in small numbers and several surviving females were able to lay eggs at the time of the following fruiting season. Implications of these findings for the ongoing geographic range expansion of the pest in temperate European countries are discussed.

Rnai-based functional analysis of bursicon genes related to cuticle pigmentation in a ladybird beetle.

In arthropods, the binding of a bursicon (encoded by burs and pburs) heterodimer or homodimer to a leucine-rich repeat-containing G protein coupled receptor LGR2 (encoded by rk) can activate many physiological processes, especially cuticle pigmentation during insect ecdysis. In the current paper, we intended to ascertain whether bursicon signaling mediates body coloration in the 28-spotted larger potato ladybird, Henosepilachna vigintioctomaculata, and if so, by which way bursicon signal governs the pigmentation. The high expression of Hvburs, Hvpburs and Hvrk occurred in the young larvae, pupae and adults, especially in the head and ventral nerve cord. RNA interference (RNAi) aided knockdown of Hvburs, Hvpburs or Hvrk in the prepupae caused similar phenotypic defects. The pigmentation of the resultant adults was affected, with significantly reduced dark areas on the sternums. Moreover, the accumulated mRNA levels of two sclerotin biosynthesis genes, aspartate 1-decarboxylase gene Hvadc and N-ß-alanyldopamine synthase gene Hvebony, were significantly increased in the Hvburs, Hvpburs or Hvrk RNAi beetles. Furthermore, depletion of either Hvadc or Hvebony could completely rescue the impaired coloration on the sternums of Hvpburs RNAi adult. Our results supported that bursicon heterodimer-mediated signal regulate cuticle pigmentation. The bursicon signaling may tune the ratio of melanins (dark/black, brown) to sclerotins (light yellow, colorless) exerting its regulative role in the pigmentation of H. vigintioctomaculata sternums.

A quantitative model of temperature-dependent diapause progression.

Winter diapause in insects is commonly terminated through cold exposure, which, like vernalization in plants, prevents development before spring arrives. Currently, quantitative understanding of the temperature dependence of diapause termination is limited, likely because diapause phenotypes are generally cryptic to human eyes. We introduce a methodology to tackle this challenge. By consecutively moving butterfly pupae of the species Pieris napi from several different cold conditions to 20 °C, we show that diapause termination proceeds as a temperature-dependent rate process, with maximal rates at relatively cold temperatures and low rates at warm and extremely cold temperatures. Further, we show that the resulting thermal reaction norm can predict P. napi diapause termination timing under variable temperatures. Last, we show that once diapause is terminated in P. napi, subsequent development follows a typical thermal performance curve, with a maximal development rate at around 31 °C and a minimum at around 2 °C. The sequence of these thermally distinct processes (diapause termination and postdiapause development) facilitates synchronous spring eclosion in nature; cold microclimates where diapause progresses quickly do not promote fast postdiapause development, allowing individuals in warmer winter microclimates to catch up, and vice versa. The unveiling of diapause termination as one temperature-dependent rate process among others promotes a parsimonious, quantitative, and predictive model, wherein winter diapause functions both as an adaptation against premature development during fall and winter and for synchrony in spring.

High-Precision Calorimetry-Based Analysis of Pupal-Pharate Adult Development in Drosophila melanogaster.

In holometabolous insects, the larval body is almost completely decomposed and reconstructed into the adult body during the pupal-pharate adult stages. Therefore, the total energetic cost of this process is a key thermodynamic quantity necessary for evaluating the benefit of their life history. Here, we measured whole-body thermal dissipation of single pupae of the fruit fly, Drosophila melanogaster, during the period from puparium formation to adult eclosion as a function of age, using a high-precision isothermal calorimeter at T = 298 K. The mass-specific energy consumption during the period from the onset of larval-pupal apolysis to adult eclosion was determined to be 2.3 kJ/g for an individual of mass (adult) = 1.0 mg, while it was observed to follow Kleiber's law for individuals smaller than mass (adult) = 1.0 mg. During the pupal-pharate adult period, in addition to the U-shaped variation, several characteristic thermal dissipations related to various events, including somatic muscle contractions, ecdyses, pulsatile hormone secretion in a pharate adult, and vaporization of the exuvial fluid, were observed. The periodic bursts in the pharate adult stage grew exponentially, suggesting that the positive feedback in the metabolic system synchronized with the progression of development, making the energy consumption in this stage more efficient. The present study showed that high-precision calorimetry is a powerful and credible method for measuring not only the total energy spent during development but also the energy spent during every specific developmental event in an organism.